Many clinical assays require multiple fluid transfers and manipulations such as addition of reactants, mixing, separation of solid and liquid phases, removal of unreacted components and undesired reaction products, and washing, etc. Oftentimes these steps have to be repeated over and over to produce the desired end result. The manipulations involved are time consuming and difficult to automate. This is a distinct disadvantage in the clinical laboratory where both time and resources are at a premium.
This problem is best exemplified in the case of immunoassays, which are techniques for determination of the presence or concentration of antigenic substances, such as those associated with a wide variety of physiological disorders, in serum or other bodily fluids. These techniques are based upon formation of a complex between the antigenic substance being assayed and an antibody or antibodies in which one or the other member of the complex may be labeled. The label, such as an enzyme or a radioactive element like I.sup.125, permits detection and/or quantitative analysis after separation of the complexed labeled antigen or antibody from uncomplexed lambed antigen or antibody.
In a sandwich immunoassay, which is one type of immunoassay technique, an antibody bound to a solid support, such as the side wall of a reaction capsule, is contacted with the fluid sample being tested. The antibody is complementary to, i.e., will complex with, a particular sought-for antigen. If present in the sample the sought-for antigen will bind to the antibody and thus the solid support. After a suitable incubation period the solid support is washed to remove the residue of the fluid sample and unreacted antigen, if any. The antibody-antigen complex on the support is next contacted with a solution containing a known quantity of labeled antibody. The labeled antibody will also complex with the sought-for antigen. After a second incubation period to promote complexing the support is again washed to remove any unreacted labeled antibody.
In a simple "yes/no" sandwich immunoassay to determine whether or not the sought-for antigen is present in the fluid sample the washed solid support is tested to detect the presence of the labeled antibody. If the label is a radioactive element, such as I.sup.125, this can be accomplished by measuring emitted radiation. The amount of labeled antibody detected is compared to that for a negative control sample known to be free of the antigen. Detection of labeled antibody in amounts significantly above the background levels demonstrated by the negative control is interpreted as indicating the presence of the sought-for antigen. Quantitative determination can be made by comparing the measure of labeled antibody with that obtained for standard samples containing known quantities of the sought-for antigen.
If the label is an enzyme, a compatible substrate, i.e., one which will be catabolized by the enzyme, is added to the reaction vessel. The action of the enzyme on the substrate may produce a change in color which would be indicative of the presence of the sought-for antigen. If quantitation is desired a substrate can be selected which, when catabolized by the enzyme, produces a measurable substance such as a fluorescing molecule. After the reaction with the enzyme has been completed the substrate is removed and the amount of fluorescing molecule generated is determined using, for example, fluorescence photometry. The concentration of the sought-for analyte may then be determined using a calibration relationship which relates the quantity of measurable substance to the quantity of the sought-for antigen in the fluid sample.
The same or similar problems of multiple fluid transfers and manipulations and repetition of steps are associated with other types of clinical assays such as infectious disease testing, therapeutic drug monitoring and hybridization probe assays.
One attempt at solving this problem with immunoassays has been to configure the solid support as a filter at the bottom of the reaction capsule. Thus, if present, the sought-for analyte will be complexed with the labeled antibody and bound to the filter. The measurable substance resulting from the action of the enzyme label on the substrate precipitates directly onto the filter leaving, for example, a colored dot on the filter. Such a colored dot is, however, difficult to quantitate with any degree of accuracy and thus such a test is, at best, a qualitative determination.
The use of a filter as a part of a reaction capsule is also known in other forms of immunoassays and, in particular, in radioimmunoassays where the filter may be of a hydrophobic material. Once the reaction within such a radioimmunoassay reaction vessel has taken place, pressure is applied to the fluid within the capsule, causing the filter to become wetted and allowing the fluid to be drawn from the reaction capsule through the filter. Once the filter is wetted, it is then not possible to terminate fluid flow through the filter. Such a characteristic of the filter material is not a disadvantage with radioimmunoassays in that such assays require fluid to be drawn from a reaction capsule through the filter only once. Were such a filter to be used in enzyme immunoassays, however, it would also be necessary to include external valves to stop fluid flow through the filter once it had been wetted. Such external valves add considerable cost and complexity to an immunoassay apparatus, particularly if the apparatus is automated and individual reaction capsules are to be discarded after a single use.